Pharmaceutical Composition and Methods for Modulating Immune System, Preventing, Pretreating and/or Treating Cancers

ABSTRACT

This invention provides a pharmaceutical composition for modulating immune system comprising at least one polysaccharide extracted from  Radix Astragali, Radix Codonopsis, Ganoderma sinense , or mixtures thereof, and their extraction process. The present invention further relates to a method of modulating immune system by applying said pharmaceutical composition to a subject suffering from cancers, and a method of preventing and/or pretreating and/or treating cancers in the subject, where said method comprises applying said pharmaceutical composition before/during chemotherapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.Non-Provisional patent application Ser. No. 14/033,528 filed Sep. 23,2013, which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 61/703,878 filed Sep. 21, 2012 and is hereby incorporated byreference in its entirety. The literatures HAN, X.-Q.; CHAN, B. C. L.;YU, H.; YANG, Y.-H.; HU, S.-Q.; KO, C.-H.; DONG, C.-X.; WONG, C.-K.;SHAW, P.-C.; FUNG, K.-P.; LEUNG, P.-C.; HSIAO, W.-L.; TU, P.-F.; HAN,Q.-B. Structural characterization and immuno-modulating activities of apolysaccharide from Ganoderma sinense. Int. J. Biol. Macromol. November2012, Vol. 51, No. 4, pages 597-603; HAN, X.-Q.; CHAN, B. C. L.; DONG,C.-X.; YANG, Y.-H.; KO, C.-H.; YUE, G. G.-L.; CHEN, D.; WONG, C.-K.;LAU, C. B.-S.; TU, P.-F.; SHAW, P.-C.; FUNG, K.-P.; LEUNG, P.-C.; HSIAO,W.-L.; HAN, Q.-B. Isolation, Structure Characterization, andImmunomodulating Activity of a Hyperbranched Polysaccharide from theFruiting Bodies of Ganoderma sinense. J. Agric. Food Chem. April 2012,Vol. 60, No. 17, pages 4276-4281; and YIN, J.-Y.; CHAN, B. C.-L.; YU,H.; LAU, I. Y.-K.; HAN, X.-Q.; CHENG, S.-W.; WONG, C.-K.; LAU, C. B.-S.XIE, M.-Y.; FUNG, K.-P.; LEUNG, P.-C.; HAN, Q.-B. Separation, structurecharacterization, conformation and immunomodulating effect of ahyperbranched heteroglycan from Radix Astragali. Carbohydrate Polymers.4 Jan. 2012, Vol. 87, No. 1, pages 667-675; CHOU TC, TALALAY P.Quantitative analysis of dose-effect relationships: the combined effectsof multiple drugs or enzyme inhibitors. Adv Enzyme Regul, 1984, 22,27-55; Z. J. JIN, Addition in drug combination. Acta Pharmacol Sin,1980, 1, 70-76 are also hereby incorporated by reference in theirentirety.

FIELD OF INVENTION

This invention provides a pharmaceutical composition for modulatingimmune system comprising at least one polysaccharide extracted fromRadix Astragali, Radix Codonopsis, Ganoderma sinense, or mixturesthereof, and their extraction process. It further relates a method ofmodulating immune system by applying said pharmaceutical composition,and a method of preventing and/or pretreating and/or treating cancerswhich comprises applying said pharmaceutical composition before/duringchemotherapy.

BACKGROUND OF INVENTION

Although great advance in the treatment of commonly seen cancers hasbeen achieved by combined use of chemotherapy, surgery, andradiotherapy, cancer therapy still faces many challenges in earlydiagnosis, metastasis, drug resistance, and notorious toxicity oftherapeutic agents to normal cells.

Combining anticancer agents with different mechanisms is a commonstrategy to improve overall efficacy; the combined use of immunotherapyand chemotherapy is one example. Nevertheless, all the agents usedthemselves are toxic and incur drug resistance; gemcitabine is a goodexample.

The medicinal herbs called Qi-tonics in Chinese medicines, like ginseng,are also used in the fight against cancers as a popular option ofalternative and complementary therapy. These herbal medicines aretraditionally used in the form of decoction and are usually rich inpolysaccharides. Many of these polysaccharides have been shown toenhance immune system and to suppress tumor growth in various animalmodels.

Chinese Medicine has a golden therapy strategy: the combined use oftonifying medicines and those having dispelling function. This strategymay fit cancer therapy right: not only to kill the cancer cells, butalso to strengthen the body's vital energy (healthy Qi) and restoreproper and healthy cell growth. A good example is Shen Qi Fu ZhengInjection (SQFZ Injection) from Livzon Group, which synergy withchemotherapy in cancer clinic has been proved in China and thereforebrought a big gross profit of HK$0.5 billion in the past half year.However, this product is facing severe technical challenge like otherChinese herbal injections, because the Chinese Government has increasedthe requirements of safety and quality control for this special kind ofmedicinal preparation.

As illustrated in FIG. 1, SQFZ Injection is derived from the waterextract of Radix Astragali and Radix Codonopsis after removal ofprecipitate in 80% ethanol. On the other hand, the removed precipitate,also called production waste, is the typical crude polysaccharidefraction, which shares the same production protocol.

Modern scientific studies have demonstrated that Astragaluspolysaccharides exhibit immuno-enhancing effects in vitro and in vivo.Chinese patent application CN1985877A discloses a composition containingRadix Astragali which strengthens immunity, prevents or treatsrespiratory tract infection, allergic rhinitis, damage of liver causedby glycogen reduction or antitumor medicine, viral hepatitis, regulatesblood viscosity and reduces blood fat.

Combined use of Radix Astragali with other herbs has been reported.China patent CN1059800C discloses an injectable composition containingginseng and Radix Astragali for treatment of immunodeficiency and immunedisorder-related diseases. Chinese patent CN1290512C discloses acomposition containing ginseng polysaccharide and Astragaluspolysaccharide as an injectable formulation for treatment of neoplasticdiseases. US patent application 2006/0110473 discloses a compositioncontaining Radix Codonopsis and Radix Astragali for preparation of animmuno-regulator and medicaments for the treatment of ischemic heartdiseases and acute lung injury. Chinese patent CN1895337B discloses acomposition containing Radix Codonopsis and Radix Astragali forprolonging mortality and improving quality of life in patients oflatter-phase cancer, and its preparation into injections, capsules andtablets.

However, knowledge about the chemistry of Astragalus polysaccharides isstill limited. It is hard to identify if the same sample was studied indifferent laboratories and it is also hard to compare theirbioactivities.

Therefore, the present invention provides a pharmaceutical compositionfor modulating immune system comprising at least one polysaccharideextracted from Radix Astragali, Radix Codonopsis, Ganoderma sinense, ormixtures thereof. While common extraction methods stated in theabovementioned prior art discard the ethanol precipitate as productionwaste, the present invention provides a process for extracting thepolysaccharides from the production waste. Also provided is a method ofmodulating immune system by applying said pharmaceutical composition anda method of preventing and/or pretreating and/or treating cancer whichcomprises applying said pharmaceutical composition before/duringchemotherapy.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art for the present application.

SUMMARY OF INVENTION

According to a first aspect of the present invention, there is provideda pharmaceutical composition for modulating immune system, comprising atleast one polysaccharide extracted from Radix Astragali, RadixCodonopsis, Ganoderma sinense, or mixtures thereof.

In a first embodiment of the first aspect, the polysaccharide has amolecular weight in the range of 500-2,500 kDa, 600-2,000 kDa, or700-1,900 kDa.

In a second embodiment of the first aspect, the polysaccharide comprisesone or more constituents selected from the group of glucose, galactose,mannose, rhamnose, arabinose, their dextrorotary and levorotarycompounds, and combinations thereof.

In a third embodiment of the first aspect, any two constituents of thepolysaccharide is in the molar ratio ranging from 1:30 to 30:1, 1:20 to20:1, or 1:10 to 10:1.

In a fourth embodiment of the first aspect, the polysaccharide comprisesglucose, galactose and mannose as constituents in the molar ratio of4.7:27.1:1.0.

In a fifth embodiment of the first aspect, the polysaccharide comprisesrhamnose, arabinose, glucose, galactose and galacturonic acid asconstituents in the molar ratio of 0.03:1.00:0.27:0.36:0.30.

In a sixth embodiment of the first aspect, the polysaccharide comprisesmainly glucose and trace amounts of galactose and mannose asconstituents.

In a seventh embodiment of the first aspect, the polysaccharide has ahyperbranched structure.

In an eighth embodiment of the first aspect, the polysaccharidecomprises at least one sugar residue selecting from the group consistingof 1,2,4-linked Rhap, α-1,4-linked Glcp, α-1,4-linked GalAp6Me,β-1,3,6-linked Galp, α-T-Araf, α-1,5-linked Araf, T-linked Araf,T-linked Glcp, T-linked Galp, (1→>6)-linked-β-D-glucopyranosyl,(1→>4)-linked-β-D-glucopyranosyl, (1→>3)-linked-β-D-glucopyranosyl,non-reducing end β-D-glucopyranosyl, t-, 1,3-, 1,4-, 1,6-, 1,3,4- and1,3,6-linked Glcp, t-linked Galp, 1,6-linked Manp, and combinationsthereof.

In a ninth embodiment of the first aspect, the polysaccharide comprisesa backbone of 1,6-linked-β-D-glucopyranosyl residues and branches at theO-3 position of every two sugar residues along the backbone.

In a tenth embodiment of the first aspect, the polysaccharide furthercomprises side chains containing 1,3-, 1,4-linked-β-D-glucopyranosyl,and non-reducing end β-D-glucopyranosyl residues.

In an eleventh embodiment of the first aspect, the polysaccharide has aprotein content of 1-20% or 5-15%.

According to a second aspect of the present invention, there is provideda process of extracting polysaccharides from Radix Astragali, RadixCodonopsis, Ganoderma sinense, which comprises:

a) Carrying out one or more solid/liquid extractions on Radix Astragali,Radix Codonopsis or Ganoderma sinense to obtain a liquid extract;b) Adding an organic solvent into the liquid extract to induceprecipitation;c) Centrifuging and filtering the mixture in (b) to obtain aprecipitate;d) Drying the precipitate to yield crude polysaccharides; ande) Purifying the crude polysaccharides by ion-exchange chromatographyand/or gel-permeation chromatography,wherein the eluents from (e) include water, bases such as sodiumhydroxide, and salt solutions such as sodium chloride solution.

According to a third aspect of the present invention, there is a methodof preventing and/or pretreating and/or treating cancers, whichcomprises applying the abovementioned pharmaceutical compositionbefore/during chemotherapy.

In a first embodiment of the third aspect, the pharmaceuticalcomposition is applied in an effective dose in the range of 0.1-20,0.5-15, 0.8-10, 1.0-5, or 1.5-3.5 mg/kg (body weight)/day.

In a second embodiment of the third aspect, the pharmaceuticalcomposition is applied in an effective dose in the range of 1.5-3.5mg/kg (body weight)/day.

In a third embodiment of the third aspect, the pharmaceuticalcomposition can be further applied simultaneously, that is jointly orseparately, or in succession, with medicaments for chemotherapy.

In a fourth embodiment of the third aspect, the medicament forchemotherapy is selected from paclitaxel, albumin-bound paclitaxel,docetaxel, or gemcitabine, or any conventional medicament forchemotherapy which has a side effect of suppressing immunity.

In a fifth embodiment of the third aspect, the pharmaceuticalcomposition is applied at the moment the subject is confirmed sufferingfrom cancers.

In a sixth embodiment of the third aspect, the pharmaceuticalcomposition is applied for at least 28 days, 21 days, 14 days, 7 days, 4days or 2 days before chemotherapy.

In a seventh embodiment of the third aspect, the pharmaceuticalcomposition is applied every day during the cycle of chemotherapy. It isa further embodiment of the present invention that this invention isapplied to a human subject.

There is a further embodiment of the third aspect that thepharmaceutical composition of the present invention is applied to thesubject being confirmed of suffering from cancers for at least 28 days,21 days, 14 days, 7 days, 4 days or 2 days before chemotherapy andduring the cycle of chemotherapy.

In yet another embodiment of the presently claimed invention, thepharmaceutical composition is applied daily while the chemotherapeuticagent for chemotherapy is applied weekly to a subject in need thereof.The daily effective dose of the pharmaceutical composition is about 3.3mg/kg while the weekly effective dose of the chemotherapeutic agent isabout 1.6 mg/kg. In that embodiment, the subject is human and route ofadministration of the chemotherapeutic agent is via intraperitoneal(i.p.) injection. In that embodiment, the pharmaceutical composition isadministered orally daily for at least a week before thechemotherapeutic agent is administered via i.p. injection on the lastday of the first week of the oral administration of the pharmaceuticalcomposition. The pharmaceutical composition of the present inventioncomprises at least one polysaccharide from Radix Astragali extract,Radix Codonopsis extract, Ganoderma sinense extract, or a mixturethereof. The chemotherapeutic agent comprises paclitaxel, albumin-boundpaclitaxel, docetaxel, and gemcitabine. The subject being administeredwith the combination of said pharmaceutical composition andchemotherapeutic agent is human who suffers from mammary cancer.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described.

The invention includes all such variation and modifications. Theinvention also includes all of the steps and features referred to orindicated in the specification, individually or collectively and any andall combinations or any two or more of the steps or features.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers. It is also noted that in this disclosure and particularly inthe claims and/or paragraphs, terms such as “comprises”, “comprised”,“comprising” and the like can have the meaning attributed to it in U.S.patent law; e.g., they can mean “includes”, “included”, “including”, andthe like; and that terms such as “consisting essentially of” and“consists essentially of” have the meaning ascribed to them in U.S.patent law, e.g., they allow for elements not explicitly recited, butexclude elements that are found in the prior art or that affect a basicor novel characteristic of the invention.

Furthermore, throughout the specification and claims, unless the contextrequires otherwise, the word “include” or variations such as “includes”or “including”, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other technical terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich the invention belongs.

Other aspects and advantages of the invention will be apparent to thoseskilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings, in which:

FIG. 1 shows the production protocol of SQFZ Injection;

FIG. 2 shows the GPC chromatogram of Example 3;

FIG. 3 shows the GPC chromatogram of Example 4;

FIG. 4 shows the GPC chromatogram of Example 5;

FIG. 5 shows (A) the ¹H NMR and (B) the ¹³C NMR spectra of Example 3;

FIG. 6 shows (A) the ¹H NMR and (B) the ¹³C NMR spectra of Example 4;

FIG. 7 shows (A) the ¹H NMR and (B) the ¹³C NMR spectra of Example 5;

FIGS. 8 a, 8 b and 8 c show the Fourier transform infrared spectra ofExample 3, 4 and 5, respectively, recorded in KBr pellets on SPECORD ina range of 400-4000 cm⁻¹;

FIGS. 9 a-9 h show in vitro effects of Example 3 on the cytokinesecretion (TNF-α, IL-1β, IL-12, IL-10, GM-CSF, IL-2, IL-4, and INF-γ,respectively) from human PBMC. PMBC (1×10⁶ cells/mL) were cultured withdifferent concentration of Example 3 (0, 40, 200, 10000, ng/mL) or LPS(8 ng/mL) for 24 hours, and the result were tested by ELISA, n=4 (4blood sample was employed in this study);

FIG. 10 shows the stimulating effect of Example 4 on the secretion ofcytokines (A) IL-1β and (B) TNF-α in PBMCs (n=8). For the assay ofcytokine secretion in PBMCs, cells were cultured with differentconcentrations of Example 4 (0.00003-100 μg/mL) for 48 h, and therelease of IL-10 and IL-12 was determined by ELISA, n=8. Dextran and LPSwere used as negative and positive control, respectively. Significantdifferences are indicated by asterisks: *, p<0.05; **, p<0.01; ***,p<0.001;

FIGS. 11 a-11 f show the cytokines production (IL-1β, IL-10, IL-2, IL-12p40, TNF-α, and GM-CSF, respectively) in PBMCs of Example 4 with theaddition of Example 5 or LPS from 2 to 10,000 ng/mL. Each bar representsthe mean±SEM of duplicates (n=7);

FIG. 12 shows the effect of Example 4 on the induction of (A) IL-10 and(B) IL-12 from human Dendritic Cell (DC);

FIG. 13 shows the effects Example 5 on (A) the cytokine levels (Upperrow from left to right: GM-CSF, IL-2, IL-5; Lower row from left toright: IL-3β, IL-10, IFN-γ) and (B) the stimulating effect on IL-6,TNF-α, IL-6 and IFN-γ in the splenocytes of the tested mice, wherein themice were divided into four groups: A. Normal; B. Tumor control; C.treatment; D. pretreatment;

FIG. 14 shows the therapeutic effect of (A) Example 1 and (B) Example 5on BALB/c mice orthotopically injected with 4T1 mouse mammary cancercells in terms of the body weight (upper left panel), spleen weight(upper right panel), tumor weight (lower left panel), and tumor/bodyweight (lower right panel) of the tumor bearing mice. The mice weredivided into four groups: A. Normal; B. Tumor control; C. treatment; D.pretreatment; and

FIG. 15 shows the therapeutic effect of different combinations ofcompounds on survival rate of a BALB/c mice model with mammary cancerorthotopically injected with 4T1 mouse mammary cancer cells over a timecourse: RAP: the pharmaceutical composition of the present invention;Taxol: paclitaxel or TAXOL®; RAP+Taxol: the pharmaceutical compositionof the present invention plus paclitaxel.

DETAILED DESCRIPTION OF INVENTION

The present invention is not to be limited in scope by any of thespecific embodiments described herein. The following embodiments arepresented for exemplification only.

In the first aspect, the invention provides a pharmaceutical compositionfor modulating the immune system comprising at least one polysaccharideextracted from Radix Astragali, Radix Codonopsis, Ganoderma sinense, ormixtures thereof. In particular, the polysaccharides have molecularweight in the range of 500-2,500 kDa, preferably 600-2,000 kDa and morepreferably 700-1,900 kDa.

The polysaccharides have hyperbranched structures and are bound to aprotein, with a protein content ranging from 1-20%, more preferably inthe range of 5-15%.

The polysaccharides comprise constituents selected from the group ofglucose, galactose, mannose, rhamnose, arabinose, their dextrorotary andlevorotary compounds, and combinations thereof.

Preferably, the polysaccharides comprise essentially glucose, galactoseand mannose as constituents with any two constituents in the molar ratioranging from 1:30 to 30:1, 1:20 to 20:1, or 1:10 to 10:1.

The polysaccharide comprises at least one sugar residue selecting fromthe group consisting of 1,2,4-linked Rhap, α-1,4-linked Glcp,α-1,4-linked GalAp6Me, β-1,3,6-linked Galp, α-T-Araf, α-1,5-linked Araf,T-linked Araf, T-linked Glcp, T-linked Galp,(1→6)-linked-β-D-glucopyranosyl, (1→>4)-linked-β-D-glucopyranosyl,(1→>3)-linked-β-D-glucopyranosyl, non-reducing end β-D-glucopyranosyl,t-, 1,3-, 1,4-, 1,6-, 1,3,4- and 1,3,6-linked Glcp, t-linked Galp,1,6-linked Manp, and combinations thereof.

In an embodiment of the invention, the polysaccharide is extracted fromGanoderma sinense. An example of the polysaccharides is defined by NMRspectra of FIGS. 5 a and 5 b, with an apparent molecular weight of 830kDa and about 6.8% protein content. Such polysaccharide presents alinear molecular structure and comprises mannose, glucose and galactoseas constituents in the molar ratio of 4.7: 27.1: 1.0. Another example ofthe polysaccharides is defined by NMR spectra of FIGS. 6 a and 6 b, withan apparent molecular weight of 1860 kDa and about 10.13% proteincontent. Such polysaccharide has a hyperbranched 1,6-glcp backbonestructure, comprising mainly glucose and trace amounts of galactose andmannose as constituents, with (1→>3)-linked glucosyl and (1→>4)-linkedglucosyl branches at O-3 position.

In another embodiment of the invention, the polysaccharides areextracted from Radix Astragali. An example of such polysaccharides has amolecular weight of 1,334 kDa and low protein content (only 0.72%). Itcomprises rhamnose, arabinose, glucose, galactose, galacturonic acid, asconstituents in a molar ratio of 0.03: 1.00: 0.27: 0.36: 0.30. Suchpolysaccharide is characterized by NMR spectra of FIGS. 7 a and 7 b.

The pharmaceutical composition according to the present invention may beoptionally added with one or more pharmaceutically acceptableauxiliaries, such as diluents, excipients, fillers, binders, wettingagents, anti-foaming agents, disintegrators, sorbefacients, surfactants,adsorption carriers, lubricants etc. These auxiliaries are known in theart and commercially available. The amount of suitable auxiliaries canbe determined by trials by those skilled in the art.

The pharmaceutical composition according to the present invention can beformulated in the form of injection, tablet, pill, capsule, powder,water-dispersible powder, granule, water-dispersible granule, paste,solution, suspension, emulsion, aerosol for inhalation. Preferable, thepharmaceutical composition is formulated as tablet, pill, capsule, andgranule.

In another aspect, the present invention provides a process ofextracting polysaccharides from Radix Astragali, Radix Codonopsis orGanoderma sinense, characterized in that one or more solid/liquidextractions are carried out. The liquid extract is precipitated with anorganic solvent, centrifuged and filtered, and then the precipitate issubsequently dried so as to yield crude polysaccharides.

The crude polysaccharides were further purified by ion-exchangechromatography followed by gel-permeation chromatography (GPC).Preferred eluents include water, bases such as sodium hydroxide, andsalt solutions such as sodium chloride solution.

In one embodiment of this method, the extraction(s) is (are) carried outwith boiling water. Preferably, the organic solvent used is ethanol.

Another aspect of the present invention is a method of modulating immunesystem by applying said pharmaceutical composition. The polysaccharidesaccording to the present invention exhibit significant immunomodulatingeffects by stimulating the proliferation of human peripheral bloodmononuclear cells (PBMCs) and enhancing its interleukin production.

The present invention also provides a pharmaceutical compositioncomprising at least one polysaccharide as defined above extracted fromRadix Astragali, Radix Codonopsis, Ganoderma sinense, or mixturesthereof, for preventing and/or pretreating and/or treating cancers.

The pharmaceutical composition according to the present invention isapplied through enteral or parenteral administration. In particular, thepharmaceutical composition is applied by injection, inhalation, oral ortopical administration. The pharmaceutical composition is preferablyapplied through oral administration.

The effective dose of the pharmaceutical composition is in the massrange of 0.1-20, 0.5-15, 0.8-10, 1.0-5, or 1.5-3.5 mg/kg (bodyweight)/day. Preferably, the mass range of effective dose of thepharmaceutical composition is 1.5-3.5 mg/kg (body weight)/day. In someembodiments, the pharmaceutical composition is applied every day. In anexemplary embodiment of the presently claimed invention, the effectivedose of the pharmaceutical composition is about 3.3 mg/kg/day.

The pharmaceutical composition is for use in preventing and/orpretreating and/or treating cancers, for example but not limited to,mammary, lung, liver, prostate, bladder, bone, bowel, colon, rectal,cervical, pancreatic, ovarian, skin, stomach, penis cancer, etc.

The pharmaceutical composition according to the present invention can befurther applied simultaneously, that is jointly or separately, or insuccession, with medicaments for chemotherapy, whereas the sequence, inthe case of separate application, generally does not have any effect onthe result of the control measures.

Suitable medicaments for chemotherapy are any chemical applied in atreatment which has a side effect of suppressing immunity. Examplesinclude paclitaxel (sold under the trademark TAXOL® by Bristol-MyersSquibb), albumin-bound paclitaxel (sold as ABRAXANE® by CelgeneCorporation), docetaxel (marketed under the name of TAXOTERE® bySanofi-Aventis), and gemcitabine (marketed as GEMZAR® by Eli Lilly andCompany).

In one embodiment, the pharmaceutical composition is applied a periodbefore chemotherapy. The application period prior to chemotherapydepends on the medicament used in chemotherapy, and target disease.Preferably, the pharmaceutical composition is applied at the moment thesubject is confirmed suffering from cancers. The pharmaceuticalcomposition is preferably applied for at least 28 days, 21 days, 14days, 7 days, 4 days or 2 days before chemotherapy.

In another embodiment, the pharmaceutical composition is applied incombination with medicaments for chemotherapy. In particular, thepharmaceutical composition is applied during the whole cycle ofchemotherapy. In some embodiments, the pharmaceutical composition isapplied every day during the cycle of chemotherapy.

In an exemplary embodiment of the presently claimed invention, thepharmaceutical composition is applied daily while the chemotherapeuticagent for chemotherapy is applied weekly to a subject in need thereof.The daily effective dose of the pharmaceutical composition is about 3.3mg/kg while the weekly effective dose of the chemotherapeutic agent isabout 1.6 mg/kg. In that embodiment, the subject is human and route ofadministration of both the pharmaceutical composition and thechemotherapeutic agent is via intraperitoneal (i.p.) injection. In thatembodiment, the pharmaceutical composition is administered via i.p.injection daily for at least a week before the chemotherapeutic agent isadministered via i.p. injection on the last day of the first week of thei.p. injection of the pharmaceutical composition, i.e., thepharmaceutical composition is administered daily for six consecutivedays before administering the chemotherapeutic agent. The pharmaceuticalcomposition of the present invention comprises at least onepolysaccharide from Radix Astragali extract, Radix Codonopsis extract,Ganoderma sinense extract, or a mixture thereof. The chemotherapeuticagent comprises paclitaxel, albumin-bound paclitaxel, docetaxel, andgemcitabine.

The following examples illustrate the invention without limiting it inany way.

Example 1

Extraction of polysaccharides from Ganoderma sinense

Dried fruiting bodies of Ganoderma sinense (1 kg) were extracted withdistilled water (12 L×2) at 100° C. for 1 hour. The combined waterextracts were concentrated to 300 mL under a reduced pressure and thencentrifuged. The supernatant was collected, to which 1.2 L of 95%ethanol was added slowly by stirring, and then kept at 4° C. overnight.The precipitate were obtained by centrifugation, then completelydissolved in appropriate volume of distilled water and intensivelydialyzed for two days (cut-off Mw 7000 Da). The retentate wasconcentrated and the protein was removed using Sevage reagent (CHCl₃:BuOH=4:1, v/v, 15 min×7). Finally, the extracts were centrifuged and thesupernatant was freeze-dried to yield the crude polysaccharides (2.7 g).

Example 2

Extraction of polysaccharides from Radix Astragali

The air-dried Radix Astragali (100 g) was cut into pieces and extractedtwice with 1.2 L boiling water for 1 hour. The solution was filtered andconcentrated under reduced pressure. The solution was precipitated withfour volumes of absolute ethanol for 12 hours. The precipitate wasresolved again in water and deproteinized using Sevag method for fivetimes. Then the solution was dialyzed against distilled water for 72hours. Finally, the retentate was lyophilized with Virtis Freeze Dryer(The VirTis Company, NewYork, USA) to yield crude polysaccharide (1.67g).

Example 3

Purification of Polysaccharide Extract by Chromatography

The crude polysaccharides (2.2 g) from Example 1 was subjected to a DEAESepharose CL-6B column (5.0×70.0 cm) and eluted first with H₂O and thenstepwise with 0.1 M, 0.3 M, 0.5 M, 1.0 M and 0.2 M sodium hydroxide(NaCl), successively, to give 6 subfractions (Fr.1-6). Fractions of 15mL were collected and monitored by phenol-H₂SO₄ method and UV absorbanceat 280 nm Each fraction was dialyzed and lyophilized. The most abundantfraction Fr.4 was further purified by gel-permeation chromatography(GPC) on Sephacryl S-300 and 400 HR (eluting with distilled water) togive the polysaccharide (615 mg). FIG. 2 shows the GPC chromatogram.

Example 4

Purification of Polysaccharide Extract by Chromatography

A portion of the crude polysaccharides (2.2 g) from Example 1 dissolvedin water (50 mL), was loaded on a DEAE-Sepharose CL-6B column (5.0×70.0cm), and eluted with a 6-step gradient with distilled water, 0.1 Msodium chloride (NaCl), 0.3 M NaCl, 0.5 M NaCl, 1.0 M NaCl, and 0.2 Msodium hydroxide (NaOH). The elution was monitored using thephenol-sulfuric acid method. The 0.2 M NaOH fraction was collected as amajor fraction, neutralized by 0.2M hydrochloric acid, dialyzed,lyophilized, and purified by gel-permeation chromatography on SephacrylS-300 and 400 HR eluting with water, to afford a purifiedpolysaccharide. FIG. 3 shows the GPC chromatogram.

Example 5

Purification of Polysaccharide Extract by Chromatography

The crude polysaccharide from Example 2 was dissolved in distilled water(4 mg/mL), filtered through a 0.45 μm membrane and separated by theBuchi Purifier system (BUCHI Labortechnik AG, Switzerland) coupled witha Hiload 26/60 Superdex-200 (2.6×60 cm) column, eluted with water at aflow rate of 2 mL/min. Fractions were collected every 3 minutes andchecked using phenol-H₂SO₄ under UV detection at 490 nm GPC was used totest the homogeneity of the purified polysaccharide. FIG. 4 shows theGPC chromatogram.

Example 6

Characterization of polysaccharides by Monosaccharide CompositionAnalysis

The polysaccharides of Example 3 and 4 (10 mg) were separatelyhydrolyzed with 2 M trifluoroacetic acid (TFA) at 100° C. for 3 hours.The monosaccharides were analyzed by GC-MS after completely convertedinto their acetylated derivation by method of Lawrence and Lyengar.Monosaccharide composition analysis indicated that polysaccharides ofExample 3 contained mannose, glucose and galactose in the molecularratio of 4.7: 27.1: 1.0, while that of Example 4 was mainly composed ofglucose, with trace amounts of galactose and mannose.

Example 7

Characterization of polysaccharide by Monosaccharide CompositionAnalysis

The polysaccharide of Example 5 was hydrolyzed with 2 M TFA at 120° C.for 2 hours in a sealed test tube. The acid was removed under reducedpressure by repeated evaporation with methanol, and then the hydrolysatewas converted into alditol acetates, based on the publication of ThomasM. Jones and Peter Albersheim, A gas chromatographic method for thedetermination of aldose and uronic acid constituents of plant cell wallpolysaccharides in Plant Physiology 1972, 49(6), pp. 926-936. ShimadzuGC/MS-QP2010 equipment (Nishinokyo Kuwabaracho, Kyoto, Japan) was usedfor the identification and quantification of monosaccharides. Thepolysaccharide of Example 5 was shown to contain rhamnose, arabinose,glucose, galactose, and galacturonic acid in the molecular ratio of0.03:1.00:0.27:0.36:0.30.

Example 8

Characterization of polysaccharide by NMR

Polysaccharides of Example 3, 4 and 5 (25 mg) were separately dried invacuum over P₂O₅ for 72 h, and then exchanged with deuterium bylyophilizing with D₂O three times. The deuterium exchangedpolysaccharide was put in a 5-mm NMR tube and dissolved in 1.0 mL 99.96%D₂O. All NMR spectra were obtained with a Bruker AM 700 spectrometerwith a dual probe in the FT mode at room temperature. TMS was used asexternal standard for the ¹³C NMR spectrum, and D₂O was used as internalstandard for ¹H NMR spectrum. FIGS. 5-7 illustrate the ¹H and ¹³C NMRspectra of the polysaccharides of Example 3, 4 and 5.

Example 9

Characterization of polysaccharide by FT-IR

The FT-IR spectra (KBr pellets) of polysaccharides of Example 3, 4 and 5were recorded on SPECORD in a range of 400-4000 cm⁻¹ and shown in FIGS.8 a-8 c.

Example 10

In vitro Immunomodulatory activity on human peripheral blood mononuclearcells (PBMC)

Immunomodulatory activities of the polysaccharides of Example 3, 4 and 5were determined by the capacity of the compounds to influence thecytokine production by human PBMC. Fresh human buffy coat obtained from6 healthy adult volunteers (Hong Kong Red Cross Blood TransfusionService) was diluted with phosphate-buffered saline at a ratio of 1:1.The diluted sample (20 mL) was put in a 50 mL centrifuge tube togetherwith an equal volume of Ficoll-Plaque Plus solution. The tube was thencentrifuged at 800 g for 20 min at 18° C. The supernatant was discardedand the PBMCs were resuspended in 4 mL RPMI 1640 medium plus 10% fetalbovine serum (FBS). The cell number was counted and the viability of thecell was checked by trypan blue exclusion assay.

The isolated PBMCs were seeded in a 96-well flat bottom microplate andincubated with the polysaccharides of Example 3, 4, 5, LPS or dextran(0.00003 to 100 μg/mL). After 24 h treatment, concentrations of IL-1βand TNF-α in culture supernatant were determined using ELISA kits (BDPharmingen Corp., Calif., USA). Polyminxin B (PMB) which is a specificinhibitor of LPS was added in the samples of polysaccharides of Example3, 4 and 5 in order to exclude the influence of LPS.

FIGS. 9-11 show the effects of the polysaccharides on the cytokineinduction from human PBMCs. The results show that the polysaccharidessignificantly stimulate the production of cytokine of human PBMCs.

Example 11

Effect on Production of IL-10 and IL-12 in Human Dendritic Cell (DC)

For generation of human monocyte derived dendritic cells (DC) from PBMC,monocytes were positively purified from PBMCs by attachment method.Cells were cultured at 2×10⁶ cells/mL in RPMI/10% FBS mediumsupplemented with granulocyte-GM-CSF (50 ng/mL) and IL-4 (40 ng/mL) for6 days. The immature DC were then harvested and incubated for 2 days byadding Example 4, LPS, or dextran (0.00003-100 μg/mL) Supernatants werecollected, and the concentrations of IL-10 and IL-12 were determined byELISA. FIG. 12 shows that the polysaccharides obtained in Example 4activate DC by stimulating the secretion of IL-12 and IL-10 from DC.

Example 12

In vivo Effect on cytokine levels on human PBMCs and mice splenocytes

In order to confirm the anti-tumor effects of the polysaccharidesobtained in Example 5, the serum levels of multiple anti-inflammatorycytokines were determined using a Bio-Plex Pro Assay mouse multiplex 6cytokine kit (Bio-Rad Laboratories, Inc., Hercules, Calif., USA), whichincluded granulocyte monocyte colony-stimulating factor, TNF-α, IL-113,IL-2, IL-10, and IFN-γ, on a Bio-Plex 200 suspension array system(Bio-Rad) equipped with a Bio-Plex manager 6.0 software.

Polysaccharides obtained in Example 5 promote cytokine production ofhuman PBMCs and mice splenocytes (FIGS. 13 a and 13 b). Compared tothose cytotoxic agents, Example 5 itself is not cytotoxic.

Example 13

Therapeutic effect on BALB/c mice orthotopically injected with 4T1 mousemammary cancer cells

Female BALB/c mice (6-8 weeks of age) were provided by Laboratory AnimalServices Center, The Chinese University of Hong Kong (CUHK), and werehoused under pathogen-free conditions, approved by AnimalExperimentation Ethics Committee of CUHK. 4T1 cells (4×10⁵),re-suspended in 0.2 ml phosphate buffered saline (PBS), weresubcutaneously (s.c.) inoculated at the mammary fat pad of each mouse.The tumor-bearing mice were randomly divided into three groups (n=10):control group (D₂O, oral fed everyday), pretreatment group, andtreatment group. Pretreatments of Example 1 and 5 (40 mg/kg/day/mouse)were initiated one week before cancer cell implantation and werecontinued every day for four weeks. For treatment group, treatments ofExample 1 and 5 (40 mg/kg/day/mouse) were initiated after cancer cellimplantation and were continued every day for four weeks. Body weight ofeach mouse was measured once a week during treatment period. At day 28,mice were sacrificed; serum samples were collected for cytokinemeasurement, and the spleens were removed for in vitro lymphocytetransformation assay.

As illustrated by FIGS. 14 a and 14 b, daily feeding with either Example1 or 5 significantly suppressed tumor growth in orthotopic 4T1 murinemammary carcinoma xenograft, without toxicity. Pretreatment with eitherExample 1 or 5 (group D) showed much better anticancer effect.

Example 14

Synergistic anticancer effect of combined use of the polysaccharidecomposition of the present invention (“RAP”) and Taxol, a knownchemotherapeutic agent, against the tumor growth in mice orthotopicallyinjected with 4T1 murine mammary cancer cells. Six- to eight-week-oldfemale BALB/c mice were divided into four groups (8-10 mice/each group):Control group and all the other three groups implanted with tumor cells(2×10⁶) at day 7; RAP group and RAP+Taxol group daily gavaged with RAP(40 mg/kg/day) since day 0; and Taxol group and RAP+Taxol group treatedwith i.p. injection of Taxol (20 mg/kg/week) since day 17.

In this example, the average body weight of mice is 20 g; and the dosesof Taxol and RAP are 20 mg/kg, and 40 mg/kg, respectively.

According to the commonly used Meeh-Rubner conversion formula, the doseratio between mice and human is approximately 12:1, so the human dosesof RAP and Taxol will be 3.3 mg/kg/day, and 1.6 mg/kg/week,respectively.

In details:

Human dose (mg/kg)=mice dose (mg/m²)×human BSA (m²)/human body weight(kg)Human BSA=0.0061×body height (cm)+0.0128×body weight (kg)−0.1529;Mice dose (mg/m²)=mice dose (mg/kg)/mice BSA (m²);

Mice BSA=9.1×(W²¹³/10000).

W represents body weight (g), BSA represents body surface area.Suppose the average human body weight is 60 kg, the height is 170 cm,so:Human BSA=0.0061×170+0.0128×60-0.1529=1.6521 m²Mice BSA=9.1×20^(2/3)/10000=0.0067 m²Mice dose (mg/m²) of Taxol=20×20×10⁻³ /0.0067=59.7 mg/m²Mice dose (mg/m²) of the RAP=40×20×10⁻³ /0.0067=119.4 mg/m²

Therefore,

Human dose (mg/kg) of Taxol will be 59.7×1.65/60=1.64 mg/kg/weekHuman dose (mg/kg) of the RAP will be 119.4×1.65/60=3.28 mg/kg/day

The results indicate that the RAP, Taxol, and RAP+Taxol exhibitsignificant anticancer effect, compared to control group. When all themice of Control group died at day 50, no death occurred in both Taxoland RAP+Taxol groups. More interestingly, the survival rate of RAP+Taxolgroup remained at 75%, while that of Taxol group dived to only 30% atDay 55. Finally, 25% of RAP+Taxol group was still alive at Day 75, whichis the end of the experiment, but all mice in other groups died earlybefore/at day 65. The Q value calculated using data of Taxol, andRAP+Taxol groups is above 1 since day 50. It is suggested that thecombined use of RAP and Taxol generates synergistic anticancer effect.

INDUSTRIAL APPLICABILITY

The present invention discloses a pharmaceutical composition formodulating immune system comprising at least one polysaccharideextracted from Radix Astragali, Radix Codonopsis, Ganoderma sinense, ormixtures thereof, and their extraction process. It further discloses amethod of modulating immune system by applying said pharmaceuticalcomposition, and a method of preventing and/or pretreating and/ortreating cancers which comprises applying said pharmaceuticalcomposition before/during chemotherapy.

If desired, the different functions discussed herein may be performed ina different order and/or concurrently with each other. Furthermore, ifdesired, one or more of the above-described functions may be optional ormay be combined.

While the foregoing invention has been described with respect to variousembodiments and examples, it is understood that other embodiments arewithin the scope of the present invention as expressed in the followingclaims and their equivalents. Moreover, the above specific examples areto be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. Without furtherelaboration, it is believed that one skilled in the art can, based onthe description herein, utilize the present invention to its fullestextent. All publications recited herein are hereby incorporated byreference in their entirety.

What is claimed is:
 1. A method of treating mammary cancer comprisingadministering an effective amount of a pharmaceutical composition incombination with at least one chemotherapeutic agent in an effectivedose to a subject in need thereof, said pharmaceutical compositioncomprising Radix Astragali extract, Radix Codonopsis extract, Gandodermasinense extract, or a mixture thereof.
 2. The method of claim 1, whereinthe mixture has a molecular weight in the range of 500-2,500 kDa,600-2,000 kDa, or 700-1,900 kDa.
 3. The method of claim 1, wherein themixture consists of a constituent selected from the group of glucose,galactose, mannose, rhamnose, arabinose, and combinations thereof. 4.The method of claim 3, wherein any two of the constituents is in themolar ratio ranging from 1:30 to 30:1, 1:20 to 20:1, or 1:10 to 10:1. 5.The method of claim 1, wherein the mixture consists of glucose,galactose and mannose as the constituents in the molar ratio of4.7:27.1:1.0.
 6. The method of claim 1, wherein the mixture consists ofrhamnose, arabinose, glucose, galactose and galacturonic acid as theconstituents in the molar ratio of 0.03:1.00:0.27:0.36:0.30.
 7. Themethod of claim 1, wherein the mixture consists mainly of glucose andtrace amounts of galactose and mannose as the constituents.
 8. Themethod of claim 1, wherein the mixture has a hyperbranched structure. 9.The method of claim 1, wherein the mixture consists of at least onesugar residue selecting from the group consisting of 1,2,4-linked Rhap,α-1,4-linked Glcp, α-1,4-linked GalAp6Me, β-1,3,6-linked Galp, α-T-Araf,α-1,5-linked Araf, T-linked Araf, T-linked Glcp, T-linked Galp,(1→>6)-linked-β-D-glucopyranosyl, (1→4)-linked-β-D-glucopyranosyl, (1→>3)-linked-β-D-glucopyranosyl, non-reducing end β-D-glucopyranosyl, t-,1,3-, 1,4-, 1,6-, 1,3,4- and 1,3,6-linked Glcp, t-linked Galp,1,6-linked Manp, and combinations thereof.
 10. The method of claim 1,wherein the mixture consists of a backbone of1,6-linked-β-D-glucopyranosyl residues and branches at the O-3 positionof every two sugar residues along the backbone.
 11. The method of claim1, wherein the mixture consists of side chains containing 1,3-,1,4-linked-β-D-glucopyranosyl, and non-reducing end β-D-glucopyranosylresidues.
 12. The method of claim 1, wherein the mixture has a proteincontent of 1-20% or 5-15%.
 13. The method of claim 1, wherein theeffective amount of said pharmaceutical composition is 0.1-20, 0.5-15,0.8-10, 1.0-5, or 1.5-3.5 mg/kg to the body weight of said subject perday and said subject is human.
 14. The method of claim 13, wherein theeffective amount of said pharmaceutical composition is 1.5-3.5 mg/kg tothe body weight of said subject per day and said subject is human. 15.The method of claim 1, wherein said pharmaceutical composition isadministered for at least 28 days, 21 days, 14 days, 7 days, 4 days or 2days before administering said chemotherapeutic agent to said subject.16. The method of claim 15, wherein the effective dose of said at leastchemotherapeutic agent is about 1.6 mg/kg/week when being administeredin combination with said pharmaceutical composition in about 3.3mg/kg/day to said subject for six consecutive days prior to theadministration of said at least chemotherapeutic agent, and said subjectis human.
 17. The method of claim 1, wherein said at least onechemotherapeutic agent comprises paclitaxel, albumin-bound paclitaxel,docetaxel, and/or gemcitabine.
 18. The method of claim 1, wherein saidat least one chemotherapeutic agent is paclitaxel.
 19. The method ofclaim 1, wherein said pharmaceutical composition is administered orallywhile said at least one chemotherapeutic agent is administered viaintraperitoneal injection to said subject.